PROJECTSUMMARY Dentalcariesisthemostprevalentchronicdiseaseinbothchildrenandadults,affecting80?90%ofthe worldpopulation3.4.Thebacterium,Streptococcusmutans,istheprimarycausativeagentofthisdiseaseandis frequentlyfoundtogetherwithStreptococcussobrinusinsituationsofmoreseveredentalcaries12,18,19.While manyofthevirulencefactorscontributingtothecariogenictyofS.mutanshavebeenwellstudied,the mechanismsthatregulatevirulencegeneexpressionhasreceivedlessattention.Understandingthe mechanismsbywhichS.mutansregulatesitsvirulencearecriticalfordevelopingtherapeuticapproachesto mitigatedentalcaries.Manybacteria,includingstreptococci,areknowntoregulategeneexpressionthrougha cell-to-cellcommunicationmechanismcalledquorumsensing(QS)21.Previousworkbyourlabhas demonstratedthatRgg-typeQSpathwaysregulatebiofilmformationandvirulenceexpressionin StreptococcuspyogenesandgeneticcompetenceinS.mutans26,35.WehaveidentifiedadditionalRgg pathwaysinS.mutansandourpreliminarydatahasidentifiedputativevirulencegenescontrolledbyeachRgg therebyprovidinginsightonthebiologicalfunctionofRggQScircuits.Moreover,thevirulenceofS.mutans mayalsobeinfluencedthroughinteractionswithS.sobrinus.Thecurrentproposalwillexplorethe mechanismsbywhichS.mutansregulatesgeneexpressionaloneandinthepresenceofS.sobrinus.The centralhypothesisofthisproposalisthatvirulencegeneexpressionbyS.mutansismodulated throughRgg-mediatedQSpathwaysandthroughitsinteractionwithS.sobrinus.Thespecificaimsof thisresearchare(1)DescriberegulatoryfunctionandchemicalsignalingforRggparalogsinS.mutans.(2) CharacterizethesynergisticeffectsofS.mutansandS.sobrinusinteraction.Toelucidatethefunctionalroles ofeachRggregulon,wewillperformaseriesofphenotypicexperiments,eachofwhichexploitsavirulence traitsuggestedbyourpreliminarydata,onrggdeletionmutants.Wewillthenanalyzethegenome-wide expressionprofileofeachmutantusingRNA-seqtodeterminegenenetworksunderpinningthephenotypes observedandtoidentifyadditionaltranscriptionaltargetsofeachRgg.TheinfluenceofS.sobrinusonS. mutans?geneexpressionwillalsobeassessed.WewillpreformRNA-seqondual-speciesbiofilmsofS. mutansandS.sobrinustodeterminetheinfluenceofthisinteractionongeneexpression.Geneticpathways showingdifferentialexpressionwillbetopcandidatesthatcontributetointerspeciesinteraction,andmutantsof thesepathwayswillbegeneratedandtestedfortheircontributiontovirulence.Itisanticipatedthatthis researchwilldelineatecriticalaspectsofS.mutanspathogenesisthatwillhelpimprovestrategiesof preventionandtreatmentofdentalcaries.Thisresearchisalsoanimportantstepinachievingthelong-term goalofdefiningtheintra-andinterspeciesQSmechanismsthatmodulatevirulenceinS.mutans,andhow theseprocessescontributetodiseasepathogenesis.